A critical step in the in vitro formation of recombinant DNA constructs and the production of genetic libraries, as well as other genetic engineering techniques, is the process of inserting DNA (and other similar polynucleotides) into host cells. The process of introducing polynucleotides into host cells is referred to as transformation. Bacterial cells are frequently used as host cells for a wide variety of genetic engineering experiments. Among the most frequently used species of bacteria for genetic engineering experiments is the organism Escherichia coli (E. coli). E. coli does not naturally import exogenous DNA into the cell. E. coli must be subjected to a process that renders it amenable to uptake of exogenous DNA, i.e., a competency inducing procedure. E. coli, as well as other bacterial cells, that have been rendered capable of taking up exogenously added DNA are referred to as competent cells.
There are many established procedures for rendering E. coli cells competent. These procedures include the CaCl.sub.2 incubation methods of Mandel and Higa, J. of Mol. Biol. 53:159 (1970), as well as numerous well-known variants thereof. Hanahan has made a detailed study of factors that effect the efficiency of transformation of E. coli cells (J. Mol. Biol. 166:557-580 (1983)) where he describes a method of producing highly competent E. coli cells comprising the step of washing E. coli cells in a buffer comprising potassium acetate, KCl, MnCl.sub.2, CaCl.sub.2, and hexamine cobalt chloride, which is generally regarded as the best available method of producing highly competent E. coli. Another method of producing competent E. coli cells is described by Jessee et al., U.S. Pat. No. 4,981,797. Jessee et al. shows that high levels of competency may be induced by growing E. coli cells in a temperature range of 18.degree. C. to 32.degree. C. as part of a competency inducing process.
The various techniques for rendering E. coli cells competent produce compositions of competent E. coli cells that vary widely in transformation efficiency. The mechanism by which DNA enters competent E. coli is not completely understood. Nor is it completely understood why one composition of competent E. coli cells differs in transformation efficiency from that of another composition of competent E. coli cells. Hanahan, in Escherichia Coli and Salmonella Typhimurium: Cellular and Molecular Biology, editor F. C. Neidhardt, American Society for Microbiology, Washington, D.C. (1987), provide a review of what is known about E. coli transformation. Only a fraction of the cells in a preparation of competent E. coli cells, i.e., a higher percentage of cells capable of being transformed, are necessarily competent for DNA uptake. Thus, some superior methods of generating competent E. coli cell compositions may simply result in the formation of competent cell compositions that contain a higher percentage of competent E. coli cells as opposed to containing E. coli cells that show individual levels of higher rates of DNA uptake or the ability to take up larger pieces of DNA. Alternatively, other methods of producing competent cells may result in the formation of competent E. coli cells that "individually" have higher transformation efficiencies. Hanahan, in J. Mol. Bio. 166:557-580 (1983) has speculated that competent E. coli cells contain channels for transport of DNA across the cell envelope and that the limiting step in determining the competency for transformation of E. coli cells are events that occur in the cell after the cell has taken up the DNA of interest, i.e., the establishment step. Another factor affecting the transformation efficiency of a composition of competent E. coli cells is the genotype of the cells. Some strains of E. coli are known to produce more highly transformable competent cell compositions than other strains of E. coli when subjected to the same competency inducing procedure. Hanahan, U.S. Pat. No. 4,851,348, describes how E. coli deoR mutants can be used to produce highly transformable E. coli cell compositions using a variety of competency inducing procedures.
Ever since it has been demonstrated that E. coli could be rendered competent to transformation, it has been of interest to produce the most competent E. coli cell preparations possible. The maximum level of transformation efficiency obtained using the method of Hanahan described in J. Mol. Bio. 166:557-580 (1987), which employs the step of washing cells in a buffer comprising potassium acetate, KCl, MnCl.sub.2, CaCl.sub.2, glycerol, and hexamine cobalt chloride, is approximately 1.times.10.sup.9 transformants per microgram of supercoiled pUC18 plasmid DNA. On a per cell basis, this translates to approximately 1 cell out of 30 in the population actually becoming transformed. However, there continues to exist a need for new and improved methods for producing competent E. coli of superior transformability, as well as new strains of E. coli that demonstrate superior transformability. Such methods and strains would be of wide interest to most researchers in the field of genetic engineering in that the number of transformations required to obtain the desired result would be minimized. Thus, for example, larger genetic libraries could be built more easily as well as the construction of complex recombinant molecules achieved more readily.